The present invention relates to a spotting device for fabricating microarrays of biological samples and a spotting pin inserted therein. More particularly, the present invention is directed to a spotting device, which comprises several pins and pin-guiding holder that allows the pins to move accurately and rectilinearly toward up-down direction by guiding and supporting pins at several points, and spotting pins inserted therein.
Since the human genome project was completed, a large amount of genetic information has been disclosed incessantly. Several techniques through which functions of genes in various organisms can be interpreted efficiently based on such information have been developed.
For the commercialization of the outcome of the human genome project, functional genomics or system biology is emerging as a subject of research in post-genome era. As a research tool these emerging fields known as a DNA chip, wherein numerous DNA fragments are arrayed on a slide glass or silicon substrate, has been commercialized. The DNA microarraying technology, what is referred to herein as the DNA chip technology, is a quite useful research tool for interpreting expression of a specific gene, mutation, polymorphism and the like, at one time. In addition, the DNA chip technology is a very suitable technique for observing the extent of expression of a known gene, or for screening out or searching for novel genes. See Lipshutz, R. J. et al. (1995) Biotechniques 19, 44 2-447; Chee, M. et al. (1996) Science 274, 610-614, the teachings of which are expressly incorporated herein by reference.
The DNA chip may be prepared through an automatized mechanical device based on molecular biological information, mechanics and electronics. In addition, the DNA chip may be prepared through the integration of numerous kinds of DNA fragments within a small area of a slide glass or silicon substrate. Therefore, the DNA chip makes it possible to screen out or search for many kinds of genes at once within a short time.
Up to now, various types of DNA chip manufacturing technology such as the spotting or contact printing method, the non-contact printing method, and the photolithography have been developed. According to the non-contact printing method, electric force generated by heat, solenoid actuator or piezoelectric actuated device causes a predetermined amount of DNA solution to drop on a plate. However, several reservoirs for the DNA solution are generally required because many kinds of DNA fragments should be integrated in a DNA chip. In addition, because the residual space in the interior of a channel buffers the pressure for dropping the DNA solution, it is difficult to drop the defined volume of the DNA solution accurately.
According to the spotting or contact-printing method, a precise pin having the shape of a sharp needle is dipped in the DNA oligomer solution, a tip of the pin contacts on a slide glass, and then a very small amount of the DNA oligomer solution print on the slide glass. However, in the case of using a needle-shaped pin having no reservoir for the DNA oligomer solution, the pin should be dipped in the solution whenever spotting. Therefore, this pin can be used only in the device that employs a kind of special arrayer or colony-picking robot.
In order to overcome this limitation, a stealth pin having about 100 micron wide grooves in the interior and the exterior of the pin has been suggested. A pin having such shape draws up the DNA oligomer solution by the capillary phenomenon when dipped in the solution, and makes it possible to print the DNA oligomer solution repeatedly on several plates. Up to now, spotting pins disclosed in U.S. Pat. Nos. 6,110,426 and 5,807,522, issued to Shalen et al. and Brown et al., respectively, and U.S. Pat. No. 6,101,946 have been known as those used in the spotting or contact-printing method issued to Martinsky. These pins have the space for reserving the sample solution. The size or the shape of the space is variable.
In use these pins can also bear impact by repetitive contact and block the reaction with the sample solution because these pins are made of stainless steel.
These pins are dipped in the sample solution and draw up the sample solution into the interior of a capillary by the capillary phenomenon. At that time, the contact between the sample solution and the inner wall of a capillary causes the difference of the surface energy. The sample solution forms a convexity or a concavity at the tip of the pins thereby. And then, the pins touch the surface of a plate, and spot the sample on the plate.
The shape of a stealth pin is known to be most suitable among existing pins for manufacturing the DNA chip by the spotting method. The shape of a stealth pin comprises a flat tip and an open-sided exterior channel reserving the predetermined volume of the sample solution.
This stealth pin is prepared through EDM (electric discharge process) and the like. According to EDM, at first one end of a metal shaft is incised as much as the predetermined length along the central axis of the pin, so that open-sided channel is formed. Finally, the gap is formed in the point. Generally, the upper part of this channel can be used for reserving the sample solution drawn through the channel by the capillary force. However, this pin can reserve only the predetermined volume of the sample solution because the enlargement of the upper part cannot but be limited. Therefore, this structure maximizes the contact between the sample solution and the inner surface of the pin and results in the high surface tension, so that a meniscus is formed concavely inward into the channel.
In addition, a tip of the pin should contact a plate with strong impact in order to spot the sample solution on a plate by using the spotting pin having the above-described shape. More specifically, a tip of the pin should contact the plate with a fixed velocity or with an accelerated velocity due to inward meniscus, in order to drop the defined volume of the reagent solution on a plate. Thereafter, movement of the pin suddenly stops and the sample solution prints on the plate. That is, in order to print the reagent solution on a plate by using a conventional spotting pin, a tip of the pin should contact the plate, and then the pin should move in the apposite direction or stop by the inertia of movement, in order to drop the sample on the plate.
Therefore, the conventional type of a spotting pin has a drawback that the diameter of a spot becomes larger because a tip is worn away by colliding with a plate. In addition, the plate or the pin can become damaged. Thus, precise mechanical operation is required in order to prevent the abrasion of the tip or the damage of the plate. Still further, the number of spots that can be made with each dipping is limited because the pin holds only a predetermined volume of the sample.
These things can cause many problems such as lack of durability, the irregularity of a spot, the spatial limitation in reserving the solution and so on. Therefore, they act as the big restrictions in the microarraying process that consequently requires many repeated spotting of various kinds of protein or nucleotide sample.
As will be recognized, in the pin-spotting method, several pins having a tip of 50 mm to 300 mm inner diameter are inserted in the holder. The pins are dipped into the DNA oligomer solution and are moved by the up-and-down and the left-and-right movement of a holder. And then, they draw and drop the DNA oligomer solution on a plate to form a spot of DNA oligomer.
That is, because the DNA oligomer solution spots on the plate by the up-and-down movement of the pin holder, a mechanical device should be controlled precisely. In addition, in order to form spots having 100 mm diameter within 1 cm2 area with high density, a pin is required to move precisely and rectilinearly.
However, in existing spotting devices used for microarraying DNA oligomer, a pin cannot move precisely and rectilinearly because it inclines in the holder. Therefore, conventional devices have the problem that a spot deviates from the target location or overlaps other spots.
In addition, the size of spots is irregular and a pin slides out of the target location by separating from a plate after contact. Therefore, it is difficult to fabricate an accurate and dense microarray by using existing spotting devices.
Therefore, this technical field requires a spotting pin i) to increase the number of spot that can be spotted through one dipping ii) to minimize the contact between its tip and a plate, iii) to minimize impact added to itself and a plate at the moment of contact, iv) to maintain the regularity of the amount and the shape of spots, and finally v) to be improved in the aspect of the durability and quality compared with existing spotting pins; and a pin spotting device to make spotting pins move accurately rectilinearly in the up-and-down direction.
Therefore, the object of the present invention is to provide the spotting device that comprises i) a pin-guiding holder to secure reproducibility of the location of spots and to allow pins to move accurately rectilinearly in the up-and-down direction, and ii) pins inserted into the spotting device. Further, objects of the present invention are to provide pins: i) to increase the number of spots that can be spotted through one dipping, ii) to minimize the contact between the tip of the pin and a plate, iii) to minimize impact added to itself and a plate at the moment of contact, iv) to maintain the regularity of the amount and the shape of spots spotted, and v) to be improved in the aspect of the durability and quality compared with existing spotting pins.